Glycosylated yghj polypeptides from enterotoxigenic escherichia coli (etec)

ABSTRACT

The present invention relates to glycosylated YghJ polypeptides from or derived from enterotoxigenic  Escherichia coli  (ETEC) that are immunogenic. In particular, the present invention relates to compositions or vaccines comprising the polypeptides and their application in immunization, vaccination, treatment and diagnosis of ETEC.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of and claims the benefit andpriority to U.S. patent application Ser. No. 15/766,209, filed on Apr.5, 2018, which is a U.S. National Phase Application of PCT InternationalApplication Number PCT/DK2016/050321, filed on Oct. 6, 2016, designatingthe United States of America and published in the English language,which is an International Application of and claims the benefit ofpriority to European Patent Application No. 15188608.2, filed on Oct. 6,2015. The disclosures of the above-referenced applications are herebyexpressly incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isSeqList-PLOUG237-001C1.txt, the date of creation of the ASCII text fileis Jun. 12, 2020, and the size of the ASCII text file is 24 KB.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to glycosylated YghJ polypeptides from orderived from enterotoxigenic Escherichia coli (ETEC) that areimmunogenic. In particular, the present invention relates tocompositions or vaccines comprising the polypeptides and theirapplication in immunization, vaccination, treatment and diagnosis ofETEC.

BACKGROUND OF THE INVENTION

Enterotoxigenic Escherichia coli (ETEC) is the major source of E. colimediated diarrhoea in humans and livestock. ETEC infections cause morethan 280 million annual episodes of diarrhoea resulting in mortalitynumbers exceeding 300,000 deaths of children under the age of fiveyears.

The significant negative health- and socio-economic impact of ETECinfection manifests itself mainly in the third world nations with poorsanitation and inadequate supplies of clean water. ETEC is a diversegroup of pathogens defined by their ability to colonize the smallintestine and secrete heat-labile and/or heat stable enterotoxins. Thecomplex pathogenicity is further attributed to the presence ofadditional bacterial virulence genes on mobile genetic elements such asplasmids and chromosomal pathogenicity islands.

Much attention has been devoted to the understanding of how ETEC andother mucosa-associated pathogens interact with host tissue duringinfection. Recent work has revealed that bacterial protein glycosylationplays an important role in mediating adhesion, colonization and invasionof host tissue.

Up until now, the known protein glycosylation repertoire of E. coli waslimited to just four proteins, all of which are surface-exposed adhesinswith functions in bacterial pathogenesis. The prototypical ETEC strainH10407 encodes two known glycoproteins, TibA and EtpA.

While the intimate coupling between protein glycosylation and bacterialpathophysiology has become apparent, the discovery of novelglycoproteins implicated in virulence is only advancing slowly. This gapof knowledge is linked to the inherent challenges associated withglycoproteomics. The analytical tools developed for enrichment ofeukaryotic O- and N-linked glycopeptides rely on a limited set ofdefined physiochemical properties, e.g. glycan hydrophilicity orspecific lectin recognition, which are relatively rare in bacteria.

Discovery and characterization of glycoproteins is further complicatedby heterogeneous glycosylation, low abundance and poor ionization ofpeptides modified with carbohydrates compared to the non-modifiedcounterpart.

Mapping of O-linked glycan moieties has proven to be a particularlychallenging task owing to the diverse nature of carbohydrate structuresavailable for protein modification in bacteria. Although methods such asperiodic acid/hydrazide glycan labelling and metabolic oligosaccharideengineering (MOE) have identified glycoproteins in a range of bacteria,these techniques present limitations in the form of low specificity forglycosylated proteins and dependence on sugar uptake and integrationinto bacterial glycoproteins, respectively.

Although they are poorly understood, bacterial glycoproteins potentiallyconstitute an important reservoir of novel therapeutic targets, whichcould be used against bacterial pathogens.

Thus, there is a great need for understanding the glycosylation patternsof proteins originating from bacteria such as ETEC, and revealing theeffect of the glycosylations on for example immunogenicity.

SUMMARY OF THE INVENTION

An object of the present invention is to provide glycosylated YghJpolypeptides that are immunogenic.

In one aspect of the present invention, the polypeptide is YghJ (alsoknown as ETEC_3241 or CBJ02741, SEQ ID NO: 1).

Another aspect of the present invention relates to the full lengthsequence of SEQ ID NO: 1, a polypeptide or polypeptide fragment of SEQID NO: 1 having at least 75% sequence identity to the full lengthsequence, or a B- or T-cell epitope of the full length sequence, whereinthe polypeptide is glycosylated at least in one position.

Still another aspect of the present invention relates to a polypeptidecomprising:

-   -   a) SEQ ID NO: 1,    -   b) a polypeptide or polypeptide fragment of SEQ ID NO: 1 having        at least 75% sequence identity to SEQ ID NO: 1, or    -   c) a B- or T-cell epitope of SEQ ID NO: 1,

wherein the polypeptide is glycosylated in at least one position.

Yet another aspect of the present invention relates to a polypeptidecomprising:

-   -   a) SEQ ID NO: 1,    -   b) a polypeptide having at least 75% sequence identity to the        full length sequence of SEQ ID NO: 1, or    -   c) a polypeptide fragment of SEQ ID NO: 1 comprising at least 5        amino acids and having at least 75% sequence identity to a        segment of SEQ ID NO: 1, said segment of SEQ ID NO:1 having the        same number of amino acids as said polypeptide fragment,

wherein the polypeptide is glycosylated in at least one position.

A further aspect of the present invention relates to an immunogeniccomposition comprising the polypeptide as described herein.

Yet another aspect of the present invention relates to a pharmaceuticalcomposition comprising the polypeptide as described herein and at leastone pharmaceutically acceptable carrier, excipient or diluent.

Another aspect of the present invention relates to the immunogeniccomposition or the pharmaceutical composition as described herein, whichis a vaccine against ETEC.

A further aspect of the present invention relates to a nucleic acidsequence encoding a polypeptide as described herein.

In a further aspect of the present invention, the polypeptide,immunogenic composition, pharmaceutical composition or vaccine asdescribed herein is for use in preventing or treating infection causedby ETEC.

In another aspect of the present invention, the polypeptide, immunogeniccomposition, pharmaceutical composition or vaccine as described hereinis for use in the preparation of a medicament for treating infectioncaused by ETEC.

Yet another aspect of the present invention relates to the polypeptide,immunogenic composition, pharmaceutical composition or vaccine asdescribed herein for use in the diagnosis of an infection caused byETEC.

A further aspect of the present invention relates to a method forimmunizing a mammal, the method comprising administering to the mammalthe immunogenic composition, pharmaceutical composition or vaccine asdescribed herein.

Another aspect of the present invention relates to a method for treatinga mammal, which is infected with ETEC comprising administering to themammal the immunogenic composition, pharmaceutical composition orvaccine as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows MALDI MS spectrum of TTVTSGGLQR (m/z=1181.59 Da) syntheticO-linked glycopeptide. FIG. 1B shows the BEMAP reaction efficientlyreplaces the carbohydrate moiety with the 2-AEP molecule and produces aphosphopeptide with the mass of 1126.64 Da. Minor traces ofbeta-eliminated as well as intact peptide can be observed (m/z=1001.62Da and 1181.59, respectively). FIG. 1C shows the AEP modified peptide isselectively enriched with TiO₂ as both the glycopeptide and thebeta-eliminated peptide is absent in the MALDI MS spectrum. FIG. 1Dshows MALDI MS peptide mass fingerprint of Tryptic digest ofheptosylated protein Ag43. Ag43 can be digested into a mixture ofheptosylated as well as unmodified peptides. Peptides marked with anasterisk indicate heptosylation. FIG. 1E shows BEMAP convertsheptosylated peptides into phosphopeptides; modified peptides areindicated. FIG. 1F shows specific TiO₂ enrichment of phosphopeptides.

FIG. 2A shows gas-phase fragmentation properties of 2-AEP tagged peptidewith either collision-induced dissociation (CID) or the CID variant,higher-energy collisional dissociation (HCD, FIG. 2B). HCD yields a morenuanced result than CID. The AEP addition substitutes a labile glycosidebond with a stronger covalent C—N bond, which greatly improves mappingof glycosylated residues by HCD fragmentation. Moreover, HCDfragmentation yields two characteristic ions (m/z=126.03 Da andm/z=138.03 Da), useful for the identification of formerly glycosylatedpeptides in complex MS/MS spectra.

The present invention will now be described in more detail in thefollowing.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have developed a novel mass spectrometry-basedtechnique, termed BEMAP, which can be employed to map O-linkedglycoproteins from theoretically any organism.

BEMAP combines a simple reaction scheme with a highly selectiveenrichment protocol to circumvent the challenges previously associatedwith bacterial glycoproteomics. The BEMAP reaction efficientlysubstitutes O-linked carbohydrate moieties with a 2-Aminoethylphosphonic acid (AEP) group, which can be selectively isolated based onits affinity for titanium dioxide.

BEMAP has been employed to map novel protein glycosylation sites in ETECstrain H10407 and the non-pathogenic E. coli K-12 MG1655. Functionalcharacterization of an H10407Δh/dE knockout strain revealed theimportance of protein glycosylation for ETEC adhesion to humanintestinal cells.

These results, together with other recent studies of bacterialglycoproteomes, highlight protein glycosylation in bacteria as anabundant, yet largely unexplored, posttranslational proteinmodification, which is central to bacterial physiology andpathophysiology.

The ETEC glycosylated proteins (polypeptides) are important inunderstanding the immunogenicity of ETEC. The glycosylated polypeptidesdisclosed herein leads to an enhanced immunogenicity compared to thesame polypeptides that are not glycosylated.

The present inventors have therefore surprisingly found that certainproteins from ETEC causes an enhanced immunogenic response due tospecific glycosylation of the YghJ proteins or fragments thereof.

Thus, an object of the present invention is to provide glycosylated YghJpolypeptides that are immunogenic.

Glycosylated Polypeptides

The term glycosylation refers to O-linked glycosylation. This is theattachment of a sugar molecule to a hydroxyl oxygen of either a Serineor Threonine side chain in a protein.

One such glycosylated polypeptide is YghJ (also known as ETEC_3241 orCBJ02741, SEQ ID NO: 1).

Therefore, one aspect of the present invention relates to the fulllength sequence of SEQ ID NO: 1, a polypeptide or polypeptide fragmentof SEQ ID NO: 1 having at least 75% sequence identity to the full lengthsequence, or a B- or T-cell epitope of the full length sequence, whereinthe polypeptide is glycosylated at least in one position. Thepolypeptides of the present invention may be synthetic or recombinant.

Another aspect of the present invention relates to a polypeptidecomprising:

-   -   a) SEQ ID NO: 1,    -   b) a polypeptide or polypeptide fragment of SEQ ID NO: 1 having        at least 75% sequence identity to SEQ ID NO: 1, or    -   c) a B- or T-cell epitope of SEQ ID NO: 1,

wherein the polypeptide is glycosylated in at least one position.

Still another aspect of the present invention relates to a polypeptidecomprising:

-   -   a) SEQ ID NO: 1,    -   b) a polypeptide having at least 75% sequence identity to the        full length sequence of SEQ ID NO: 1, or    -   c) a polypeptide fragment of SEQ ID NO: 1 comprising at least 5        amino acids and having at least 75% sequence identity to a        segment of SEQ ID NO: 1, said segment of SEQ ID NO:1 having the        same number of amino acids as said polypeptide fragment,

wherein the polypeptide is glycosylated in at least one position.

The polypeptide fragments of the present invention may comprise at least5 amino acids, such as at least 7 amino acids, at least 8 amino acids,at least 9 amino acids, at least 10 amino acids, at least 12 aminoacids, at least 15 amino acids, at least 20 amino acids, at least 25amino acids, at least 30 amino acids, at least 35 amino acids, at least40 amino acids, at least 45 amino acids, or at least 50 amino acids.

Thus, one embodiment of the present invention relates to polypeptides asdescribed herein, wherein the polypeptide fragment comprises at least 7amino acids, such as at least 8 amino acids, at least 9 amino acids, atleast 10 amino acids, at least 12 amino acids, at least 15 amino acids,at least 20 amino acids, at least 25 amino acids, at least 30 aminoacids, at least 35 amino acids, at least 40 amino acids, at least 45amino acids, or at least 50 amino acids.

Another embodiment of the present invention relates to polypeptides asdescribed herein, wherein the polypeptide fragment comprises at least 9amino acids.

Still another embodiment of the present invention relates topolypeptides as described herein, wherein the polypeptide fragmentcomprises at least 10 amino acids.

A further embodiment of the present invention relates to polypeptides asdescribed herein, wherein the polypeptide fragment comprises at least 20amino acids.

The polypeptides of the present invention may be glycosylated at leastin two positions, such as at least in three positions, at least fourpositions, at least five positions, at least six positions, seven, eightor at least nine positions.

In one embodiment of the present invention, the polypeptide isglycosylated in at least two positions.

In another embodiment of the present invention, the polypeptide isglycosylated in at least three positions.

The polypeptides can also be glycosylated in exactly one, two, three,four, five, six, seven, eight or nine positions.

Numerous examples are known in which proteins can be extensivelyglycosylated. Glycosylated proteins can exhibit completely differentbiological functions than their non-glycosylated counterparts. In thepresent context, a hyperglycosylated protein (or polypeptide) is definedas an amino acid sequence being glycosylated in at least ten positions.

Thus, yet another embodiment of the present invention relates to thesituation, wherein the polypeptide as described herein ishyperglycosylated.

The polypeptides of the present invention may also be characterized bycertain amino acid motifs. Such motifs can be identified experimentally,by for instance BEMAP as described herein or computationally by softwaretools such as Motif-X, which recognizes overrepresented patterns from asequence data set (M. F. Chou and D. Schwartz (2011).

An embodiment of the present invention consequently relates to apolypeptide as described herein, wherein the glycosylated polypeptidecomprises at least one 35 asparagine within seven amino acids from eachglycosylated amino acid.

Therefore, embodiments of the present invention also encompassesglycosylated polypeptide comprising at least one asparagine within sevenamino acids from each glycosylated amino acid, such as within seven,six, five, four, three, two or one amino acid from each glycosylatedamino acid.

The polypeptides may also be defined by more specific amino acid motifs.A bioinformatics motif analysis of the YghJ sequence revealed severalfrequently occurring amino acid motifs, below presented by the specificamino acids as well as by X, which signifies an arbitrarily chosen aminoacid.

Motif 1: XTXNX

Motif 2: XTXXXNX

Motif 3: XTXXXXXXNX

Motif 4: XTTX

Motif 5: XSNX

Motif 6: XSXNX

Motif 7: XSTX

Motif 8: XNXXXXXXSX

Motif 9: XSXXTX

Motif 10: XSXXNX

Motif 11: XNSX

Motif 12: XXXXXXXXCSXXXXXXXXX

Motif 13: XXXXXXXXXSCXXXXXXXX

Motif 14: XXXXXXXCXSXXXXXXXXX

Motif 15: XXXXXXXXXSXCXXXXXXX

Motif 16: XXXXXXCXXSXXXXXXXXX

Motif 17: XXXXXXXXXSXXXCXXXXX

Motif 18: XXXXXCXXXSXXXXXXXXX

Motif 19: XXXXXXXXXSXXCXXXXXX

Motif 20: XXXXXXXXXSXXXXCXXXX

Motif 21: XXXXXXXXXSXXXXXCXXX

Motif 22: XXXXXXXXXTCXXXXXXXX

Motif 23: XXXXXXXXCTXXXXXXXXX

Motif 24: XXXXXXXXXTXCXXXXXXX

Motif 25: XXXXXXXCXTXXXXXXXXX

Motif 26: XXXXXXCXXTXXXXXXXXX

Motif 27: XXXXXXXXXTXXCXXXXXX

Motif 28: XXXXXXXXXTXXXCXXXXX

Motif 29: XXXXXCXXXTXXXXXXXXX

Motif 30: XXXXXXXXXTXXXXCXXXX

Motif 31: XXXXXXXXNTXXXXXXXXX

Motif 32: XXXXXXXNXTXXXXXXXXX

Thus, another embodiment of the present invention relates to apolypeptide as described herein, wherein the glycosylated polypeptidecomprises an amino acid motif selected from the group consisting ofXTXNX, XTXXXNX, XTXXXXXXNX, XTTX, XSNX, XSXNX, XSTX, XNXXXXXXSX, XSXXTX,XSXXNX, and XNSX.

In another embodiment of the present invention relates to a polypeptideas described herein, wherein the glycosylated polypeptide comprises anamino acid motif selected from the group consisting ofXXXXXXXXCSXXXXXXXXX, XXXXXXXXXSCXXXXXXXX, XXXXXXXCXSXXXXXXXXX,XXXXXXXXXSXCXXXXXXX, XXXXXXCXXSXXXXXXXXX, XXXXXXXXXSXXXCXXXXX,XXXXXCXXXSXXXXXXXXX, XXXXXXXXXSXXCXXXXXX, XXXXXXXXXSXXXXCXXXX,XXXXXXXXXSXXXXXCXXX, XXXXXXXXXTCXXXXXXXX, XXXXXXXXCTXXXXXXXXX,XXXXXXXXXTXCXXXXXXX, XXXXXXXCXTXXXXXXXXX, XXXXXXCXXTXXXXXXXXX,XXXXXXXXXTXXCXXXXXX, XXXXXXXXXTXXXCXXXXX, XXXXXCXXXTXXXXXXXXX,XXXXXXXXXTXXXXCXXXX, XXXXXXXXNTXXXXXXXXX and XXXXXXXNXTXXXXXXXXX.

In a further embodiment of the present invention relates to apolypeptide as described herein, wherein the glycosylated polypeptidecomprises an amino acid motif selected from the group consisting ofXTXNX, XTXXXNX, XTXXXXXXNX, XTTX, XSNX, XSXNX, XSTX, XNXXXXXXSX, XSXXTX,XSXXNX, XNSX, XXXXXXXXCSXXXXXXXXX, XXXXXXXXXSCXXXXXXXX,XXXXXXXCXSXXXXXXXXX, XXXXXXXXXSXCXXXXXXX, XXXXXXCXXSXXXXXXXXX,XXXXXXXXXSXXXCXXXXX, XXXXXCXXXSXXXXXXXXX, XXXXXXXXXSXXCXXXXXX,XXXXXXXXXSXXXXCXXXX, XXXXXXXXXSXXXXXCXXX, XXXXXXXXXTCXXXXXXXX,XXXXXXXXCTXXXXXXXXX, XXXXXXXXXTXCXXXXXXX, XXXXXXXCXTXXXXXXXXX,XXXXXXCXXTXXXXXXXXX, XXXXXXXXXTXXCXXXXXX, XXXXXXXXXTXXXCXXXXX,XXXXXCXXXTXXXXXXXXX, XXXXXXXXXTXXXXCXXXX, XXXXXXXXNTXXXXXXXXX andXXXXXXXNXTXXXXXXXXX.

Sequence Identity

Glycosylated polypeptides may be obtained directly from a bacterialculture by purification or they can be chemically synthesized.

In an embodiment of the present invention, the polypeptide originatesfrom Enterotoxigenic Escherichia coli (ETEC). Examples of suchpolypeptides are given in the present disclosure.

The polypeptides can also be functional variants of the polypeptidesdisclosed herein. Such variance can be determined by sequence identity.

The term “sequence identity” indicates a quantitative measure of thedegree of homology between two amino acid sequences of substantiallyequal length or between two nucleic acid sequences of substantiallyequal length. The two sequences to be compared must be aligned to bestpossible fit with the insertion of gaps or alternatively, truncation atthe ends of the protein sequences. The sequence identity can becalculated as

$\frac{\left( {N_{ref} - N_{dif}} \right)100}{N_{ref}},$

wherein N_(dif) is the total number of non-identical residues in the twosequences when aligned and wherein N_(ref) is the number of residues inone of the sequences. Hence, the DNA sequence AGTCAGTC will have asequence identity of 75% with the sequence AATCAATC (N_(dif)=2 andN_(ref)=8). A gap is counted as non-identity of the specific residue(s),i.e. the DNA sequence AGTGTC will have a sequence identity of 75% withthe DNA sequence AGTCAGTC (N_(dif)=2 and N_(ref)=8). Sequence identitycan alternatively be calculated by the BLAST program e.g. the BLASTPprogram (W. R Pearson and D. J. Lipman (1988)). In one embodiment of theinvention, alignment is performed with the sequence alignment methodClustalW with default parameters as described by J. D. Thompson et al(1994).

For calculations of sequence identity when comparing polypeptidefragments with longer amino acid sequences, the polypeptide fragment isaligned with a segment of the longer amino acid sequence. Thepolypeptide fragment and the segment of the longer amino acid sequencemay be of substantially equal length. Thus, the polypeptide fragment andthe segment of the longer amino acid sequence may be of equal length.After alignment of the polypeptide fragment with the segment of thelonger amino acid sequence, the sequence identity is computed asdescribed above.

A preferred minimum percentage of sequence identity is at least 75%,such as at least 80%, such as at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, and at least 99.5%.

Thus, one embodiment of the present invention relates to a polypeptideas described herein, wherein the polypeptide or polypeptide fragment hasat least 80% sequence identity to the full-length sequence of SEQ ID No:1, such as at least 80%, such as at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or at least 99.5%.

An embodiment of the present invention relates to a polypeptide asdescribed herein, wherein the polypeptide or polypeptide fragment has atleast 90% sequence identity to SEQ ID NO: 1.

Another embodiment of the present invention relates to a polypeptide asdescribed herein, wherein the polypeptide has at least 90% sequenceidentity to the full length sequence of SEQ ID NO: 1, and thepolypeptide fragment has at least 90% sequence identity to a segment ofSEQ ID NO: 1, said segment of SEQ ID NO:1 having the same number ofamino acids as said polypeptide fragment.

Preferably, the numbers of substitutions, insertions, additions ordeletions of one or more amino acid residues in the polypeptide islimited, i.e. no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions,no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 insertions, no more than 1,2, 3, 4, 5, 6, 7, 8, 9, 10 additions, and no more than 1, 2, 3, 4, 5, 6,7, 8, 9, 10 deletions compared to the immunogenic polypeptide unitsbased on polypeptides disclosed herein.

B- or T-Cell Epitopes

Polypeptides such as the ETEC proteins disclosed herein can containimmunogenic parts, such as B- or T-cell epitopes.

The immunogenic part of an immunogenic polypeptide is the part of thepolypeptide, which elicits an immune response in an animal or a humanbeing, and/or in a biological sample determined by any of the biologicalassays known to the skilled person working with immune responses.

The immunogenic part of a polypeptide may be a T-cell epitope or aB-cell epitope and can be related to one or a few relatively small partsof the polypeptide, they can be scattered throughout the polypeptidesequence or be situated in specific parts of the polypeptide.

In order to identify relevant T-cell epitopes which are recognizedduring an immune response, it is possible to use a “brute force” method:Since T-cell epitopes are linear, deletion mutants of the polypeptidewill, if constructed systematically, reveal what regions of thepolypeptide are essential in immune recognition, e.g. by subjectingthese deletion mutants e.g. to assays known to the skilled personworking with immune responses.

Another method utilizes overlapping oligopeptides for the detection ofMHC class II epitopes, preferably synthetic, having a length of e.g. 20amino acid residues derived from the polypeptide. These peptides can betested in biological assays and some of these will give a positiveresponse (and thereby be immunogenic) as evidence for the presence of aT cell epitope in the peptide.

For the detection of MHC class I epitopes it is possible to predictpeptides that will bind and hereafter produce these peptidessynthetically and test them in relevant biological assays. The peptidespreferably having a length of e.g. 8 to 20 amino acid residues derivedfrom the polypeptide. B-cell epitopes can be determined by analyzing theB-cell recognition to overlapping peptides covering the polypeptide ofinterest.

B-cell epitopes differ from T-cell epitopes in that they areconformational epitopes that require a three dimensional structure inorder to raise an immune response. Without being bound by theory,variants of B-cell epitopes can be identified through key amino acids(for example glycosylated amino acids) and a certain length of thepolypeptide while remaining immunogenic.

Thus, an embodiment of the present invention therefore relates toepitopes, such as B- or T-cell epitopes of the polypeptides mentionedherein.

A common feature of the polypeptides of the present invention is theircapability to induce an immunological response as illustrated in theexamples. It is understood that within the scope of the presentinvention are variants of the polypeptides of the invention produced bysubstitution, insertion, addition or deletion while remainingimmunogenic.

Examples of such epitopes are listed in the examples of the presentdisclosure and include SEQ ID NOs: 2-23. Other examples include SEQ IDNOs: 24-50. Also polypeptides with a minimum percentage of sequenceidentity to any of SEQ ID NOs: 2-50 form part of the invention.

A preferred minimum percentage of sequence identity to any of SEQ IDNOs: 2-50 is at least 75%, such as at least 80%, such as at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, andat least 99.5%.

Therefore, an embodiment of the present invention relates to apolypeptide as described herein, wherein the polypeptide has at least75% sequence identity to the full-length sequence of SEQ ID NOs: 2-50,such as at least 80%, such as at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5%.

Thus, an embodiment of the present invention relates to a polypeptide asdescribed herein, wherein the glycosylated polypeptide is selected fromthe group of glycosylated polypeptides consisting of SEQ ID NOs: 2-50and polypeptides having at least 75% sequence identity to the fulllength sequence of SEQ ID Nos: 2-50.

Another embodiment of the present invention relates to a polypeptide asdescribed herein, wherein the glycosylated polypeptide is selected fromthe group of glycosylated polypeptides consisting of SEQ ID NOs: 2-23and polypeptides having at least 75% sequence identity to the fulllength sequence of SEQ ID Nos: 2-23.

Yet another embodiment of the present invention relates to a polypeptideas described herein, wherein the glycosylated polypeptide is selectedfrom the group of glycosylated polypeptides consisting of SEQ ID NOs:24-50 and polypeptides having at least 75% sequence identity to the fulllength sequence of SEQ ID Nos: 24-50.

A further embodiment of the present invention relates to a polypeptideas described herein, wherein the glycosylated polypeptide has at least90% sequence identity to the full length sequence of SEQ ID Nos: 2-50.

An even further embodiment of the present invention relates to apolypeptide as described herein, wherein the glycosylated polypeptidehas at least 90% sequence identity to the full length sequence of SEQ IDNos: 2-23.

Still another embodiment of the present invention relates to apolypeptide as described herein, wherein the glycosylated polypeptidehas at least 90% sequence identity to the full length sequence of SEQ IDNos: 24-50.

The polypeptides may also be given by a specific sequence selected fromSEQ ID Nos: 2-50.

Therefore, an embodiment of the present invention relates to apolypeptide as described herein, wherein the glycosylated polypeptide isselected from the group of glycosylated polypeptides consisting of SEQID NOs: 2-50.

Thus, in one embodiment of the present invention, the glycosylatedpolypeptide is selected from the group of glycosylated polypeptidesconsisting of SEQ ID NOs: 2-23.

Another embodiment of the present invention relates to a polypeptide asdescribed herein, wherein the glycosylated polypeptide is selected fromthe group of glycosylated polypeptides consisting of SEQ ID NOs: 24-50.

Key features of these epitopes or fragments are that they comprise oneor more glycosylations at central positions.

Polypeptide Purity

In the present context the term “substantially pure polypeptide” means apolypeptide preparation which contains at most 5% by weight of otherpolypeptide material with which it is associated natively or duringrecombinant or synthetic production (lower percentages of otherpolypeptide material are preferred, e.g. at most 4%, at most 3%, at most2%, at most 1%, and at most ½%).

It is preferred that the substantially pure polypeptide is at least 96%pure, i.e. that the polypeptide constitutes at least 96% by weight oftotal polypeptide material present in the preparation, and higherpercentages are preferred, such as at least 97%, at least 98%, at least99%, at least 99.25%, at least 99.5%, and at least 99.75%. It isespecially preferred that the polypeptide is in “essentially pure form”,i.e. that the polypeptide is essentially free of any other antigen withwhich it is natively associated, i.e. free of any other antigen frombacteria. This can be accomplished by preparing the polypeptide by meansof recombinant methods in a host cell, or by synthesizing thepolypeptide by the well-known methods of solid or liquid phase peptidesynthesis, and by using appropriate purification procedures well knownto the person of ordinary skill in the art.

Thus in one embodiment of the present invention are the polypeptides ofthe present invention substantially pure or in essentially pure form.

Fusion Polypeptides

Two or more of the polypeptides disclosed herein may be fused to formfusion polypeptides.

Therefore, an embodiment of the present invention relates to a situationwherein the polypeptide is a fusion polypeptide.

The polypeptides to which fusion is made may originate from ETEC oralternatively be other polypeptides that are beneficial when an enhancedimmune response against ETEC is required.

Thus, another embodiment of the present invention relates to apolypeptide as described herein, wherein the polypeptide is fused to apolypeptide originating from ETEC.

Yet another embodiment of the present invention relates to a polypeptideas described herein, wherein the polypeptide is a fusion polypeptide,said fusion polypeptide comprising one or more glycosylated polypeptideselected from the group of glycosylated polypeptides consisting of SEQID NOs: 2-50.

A further embodiment of the present invention relates to a polypeptideas described herein, wherein the polypeptide is a fusion polypeptide,said fusion polypeptide comprising one or more glycosylated polypeptideselected from the group of glycosylated polypeptides consisting of SEQID NOs: 2-23.

An even further embodiment of the present invention relates to apolypeptide as described herein, wherein the polypeptide is a fusionpolypeptide, said fusion polypeptide comprising one or more glycosylatedpolypeptide selected from the group of glycosylated polypeptidesconsisting of SEQ ID NOs: 24-50.

Another embodiment of the present invention relates to a polypeptide asdescribed herein, wherein the polypeptide is a fusion polypeptide, saidfusion polypeptide consisting of two or more glycosylated polypeptidesselected from the group of glycosylated polypeptides consisting of SEQID NOs: 2-50.

Another embodiment of the present invention relates to a polypeptide asdescribed herein, wherein the polypeptide is a fusion polypeptide, saidfusion polypeptide consisting of two or more glycosylated polypeptidesselected from the group of glycosylated polypeptides consisting of SEQID NOs: 2-23.

Another embodiment of the present invention relates to a polypeptide asdescribed herein, wherein the polypeptide is a fusion polypeptide, saidfusion polypeptide consisting of two or more glycosylated polypeptidesselected from the group of glycosylated polypeptides consisting of SEQID NOs: 24-50.

Immunogenicity

An immunogenic polypeptide is defined as a polypeptide that induces animmune response. The immune response may be monitored by one of thefollowing methods:

An in vitro cellular response is determined by release of a relevantcytokine such as IFN-γ, from lymphocytes withdrawn from an animal orhuman currently or previously infected with ETEC, or by detection ofproliferation of these T cells. The induction is performed by additionof the polypeptide or the immunogenic part to a suspension comprisingfrom 1×10⁵ cells to 3×10⁵ cells per well. The cells are isolated fromeither blood, the spleen, the liver or the lung and the addition of thepolypeptide or the immunogenic part of the polypeptide result in aconcentration of not more than 20 μg per ml suspension and thestimulation is performed from two to five days. For monitoring cellproliferation, the cells are pulsed with radioactive labeled Thymidineand after 16-22 hours of incubation, the proliferation is detected byliquid scintillation counting. A positive response is a response morethan background plus two standard deviations. The release of IFN-γ canbe determined by the ELISA method, which is well known to a personskilled in the art. A positive response is a response more thanbackground plus two standard deviations. Other cytokines than IFN-γcould be relevant when monitoring an immunological response to thepolypeptide, such as IL-12, TNF-α, IL-4, IL-5, IL-10, IL-6, TGF-β.

Another and more sensitive method for determining the presence of acytokine (e.g. IFN-γ) is the ELISPOT method where the cells isolatedfrom either the blood, the spleen, the liver or the lung are diluted toa concentration of preferable of 1 to 4×10⁶ cells/ml and incubated for18-22 hrs in the presence of the polypeptide or the immunogenic part ofthe polypeptide resulting in a concentration of not more than 20 μg perml. The cell suspensions are hereafter diluted to 1 to 2×10⁶/ml andtransferred to Maxisorp plates coated with anti-IFN-γ and incubated forpreferably 4 to 16 hours. The IFN-γ producing cells are determined bythe use of labelled secondary anti-IFN-antibody and a relevant substrategiving rise to spots, which can be enumerated using a dissectionmicroscope. It is also a possibility to determine the presence of mRNAcoding for the relevant cytokine by the use of the PCR technique.Usually one or more cytokines will be measured utilizing for example thePCR, ELISPOT or ELISA. It will be appreciated by a person skilled in theart that a significant increase or decrease in the amount of any ofthese cytokines induced by a specific polypeptide can be used inevaluation of the immunological activity of the polypeptide.

An in vitro cellular response may also be determined by the use of Tcell lines derived from an immune individual or an ETEC infected personwhere the T cell lines have been driven with either live ETEC, extractsfrom the bacterial cell or culture filtrate for 10 to 20 days with theaddition of IL-2. The induction is performed by addition of not morethan 20 μg polypeptide per ml suspension to the T cell lines containingfrom 1×10⁵ cells to 3×10⁵ cells per well and incubation is performedfrom two to six days. The induction of IFN-γ or release of anotherrelevant cytokine is detected by ELISA. The stimulation of T cells canalso be monitored by detecting cell proliferation using radioactivelylabeled Thymidine as described above. For both assays, a positiveresponse is a response more than background plus two standarddeviations.

An in vivo cellular response may be determined as a positive DTHresponse after intradermal injection or local application patch of atmost 100 μg of the polypeptide or the immunogenic part to an individualwho is clinically or subclinically infected with ETEC, a positiveresponse having a diameter of at least 5 mm 72-96 hours after theinjection or application.

An in vitro humoral response is determined by a specific antibodyresponse in an immune or infected individual. The presence of antibodiesmay be determined by an ELISA technique or a Western blot where thepolypeptide or the immunogenic part is absorbed to either anitrocellulose membrane or a polystyrene surface. The serum ispreferably diluted in PBS from 1:10 to 1:100 and added to the absorbedpolypeptide and the incubation being performed from 1 to 12 hours. Bythe use of labeled secondary antibodies the presence of specificantibodies can be determined by measuring the presence or absence of aspecific label e.g. by ELISA where a positive response is a response ofmore than background plus two standard deviations or alternatively avisual response in a Western blot.

Another relevant parameter is measurement of the protection in animalmodels induced after vaccination with the polypeptide in an adjuvant orafter DNA vaccination. Suitable animal models include primates, guineapigs or mice, which are challenged with an infection of an ETEC. Readoutfor induced protection could be decrease of the bacterial load in targetorgans compared to non-vaccinated animals, prolonged survival timescompared to non-vaccinated animals and diminished weight loss orpathology compared to non-vaccinated animals.

Thus, the glycosylated polypeptides described herein are immunogenicwhen one of the above-described tests is positive.

In one aspect of the present invention are the polypeptides describedherein immunogenic.

Such an immunogenic polypeptide may be used for immunizing a subject toinfectious bacteria. Thus, an embodiment of the present inventionrelates to a polypeptide as described herein for use in immunizing amammal against ETEC.

Another embodiment of the present invention relates to a polypeptide asdescribed herein for use in immunizing a human against ETEC.

Another aspect of the present invention relates to a compositioncomprising a polypeptide as described herein. Such composition willconstitute an immunogenic composition.

Antibodies

The glycosylated polypeptides disclosed herein can constitute epitopes.

An epitope, also known as antigenic determinant, is the part of anantigen that is recognized by the immune system, specifically byantibodies, B cells, or T cells.

The epitopes of protein antigens are divided into two categories,conformational epitopes and linear epitopes, based on their structureand interaction with the paratope.

A conformational epitope is composed of discontinuous sections of theantigen's amino acid sequence.

These epitopes interact with the paratope based on the 3-D surfacefeatures and shape or tertiary structure of the antigen.

By contrast, linear epitopes interact with the paratope based on theirprimary structure. A linear epitope is formed by a continuous sequenceof amino acids from the antigen.

Thus, one aspect of the present invention relates to an antibody thatbinds to an epitope described herein.

Antibodies raised against the epitope may be either polyclonal ormonoclonal.

The antibodies may be suitable to generate chimeric and/or humanversions that could be appropriate for human in vivo use.

Thus, the invention is also concerned with the polypeptides as describedherein for use in animals to produce antisera for diagnostic andtherapeutic purposes.

Antibodies obtained from animals exposed to the polypeptides asdescribed herein, may be used for the treatment or diagnosis of abacterial infection, such as an ETEC infection.

The immunoglobulin heavy chain (IgH) is the large polypeptide subunit ofan antibody (immunoglobulin). A typical antibody is composed of twoimmunoglobulin (Ig) heavy chains and two Ig light chains.

Several different types of heavy chain exist that define the class orisotype of an antibody. These heavy chain types vary between differentanimals.

The immunoglobulin light chain is the small polypeptide subunit of anantibody (immunoglobulin).

There are two types of light chain in humans (as in other mammals),kappa (κ) chain, encoded by the immunoglobulin kappa locus on chromosome2 and the lambda (A) chain, encoded by the immunoglobulin lambda locuson chromosome 22.

Antibodies are produced by B lymphocytes, each expressing only one classof light chain.

Once set, light chain class remains fixed for the life of the Blymphocyte.

In a healthy individual, the total kappa to lambda ratio is roughly 2:1in serum (measuring intact whole antibodies) or 1:1.5 if measuring freelight chains, with a highly divergent ratio indicative of neoplasm.

The exact normal ratio of kappa to lambda ranges from 0.26 to 1.65.

Both the kappa and the lambda chains can increase proportionately,maintaining a normal ratio.

Carriers, Excipients and Diluents

Pharmaceutical compositions comprising the polypeptides described hereinmay be administered in a physiologically acceptable medium (e.g.,deionized water, phosphate buffered saline (PBS), saline, aqueousethanol or other alcohol, plasma, proteinaceous solutions, mannitol,aqueous glucose, vegetable oil, or the like).

Thus, an embodiment of the present invention relates to a compositioncomprising a polypeptide as described herein that constitutes apharmaceutical composition.

Buffers may also be included, particularly where the media are generallybuffered at a pH in the range of about 5 to 10, where the buffer willgenerally range in concentration from about 50 to 250 mM salt, where theconcentration of salt will generally range from about 5 to 500 mM,physiologically acceptable stabilizers, and the like.

The compounds may be lyophilized for convenient storage and transport.

Thus, in a further embodiment of the present invention the compositioncomprises one or more excipients, diluents and/or carriers.

Aqueous suspensions may contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions.

Such excipients include suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.

Thus, an aspect of the present invention relates to a pharmaceuticalcomposition comprising a polypeptide as described herein and at leastone pharmaceutically acceptable carrier, excipient or diluent.

Vaccines, Treatment and Administration

The polypeptides, immunogenic compositions, and pharmaceuticalcomposition may constitute a vaccine against ETEC.

Therefore, an aspect of the present invention relates to an immunogeniccomposition or a pharmaceutical composition as defined herein, which isa vaccine against ETEC.

An embodiment of the present invention relates to a polypeptide asdescribed herein for use in a vaccine against ETEC. Such a vaccine maybe for use in a mammal, preferably a human.

Another embodiment of the present invention relates to a polypeptide asdescribed herein for use in the preparation of a vaccine against ETEC.Such a vaccine may be for use in a mammal, preferably a human.

Key features of vaccines is that they are recognized by the recipient'simmune response, generate a response, and ultimately decrease thebacterial load of ETEC.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be prophylactic ortherapeutically effective and immunogenic. The quantity to beadministered depends on the subject to be treated, including, e.g., thecapacity of the individual's immune system to mount an immune response,and the degree of protection desired. Suitable dosage ranges are of theorder of several hundred micrograms of the fusion polypeptide of theinvention per vaccination with a preferred range from about 0.1 μg to1000 μg, such as in the range from about 1 μg to 300 μg, and especiallyin the range from about 10 μg to 100 μg. Suitable regimens for initialadministration and booster shots are also variable but are typified byan initial administration followed by subsequent inoculations or otheradministrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These includeoral, nasal or mucosal application in either a solid form containing theactive ingredients (such as a pill, suppository or capsule) or in aphysiologically acceptable dispersion, such as a spray, powder orliquid, or parenterally, by injection, for example, subcutaneously,intradermally or intramuscularly or transdermally applied. The dosage ofthe vaccine will depend on the route of administration and will varyaccording to the age of the person to be vaccinated and, to a lesserdegree, the size of the person to be vaccinated. Currently, mostvaccines are administered intramuscularly by needle injection and thisis likely to continue as the standard route. However, vaccineformulations that induce mucosal immunity have been developed, typicallyby oral or nasal delivery. One of the most widely studied deliverysystems for induction of mucosal immunity contains cholera toxin (CT) orits B subunit. This protein enhances mucosal immune responses andinduces IgA production when administered in vaccine formulations. Anadvantage is the ease of delivery of oral or nasal vaccines. Modifiedtoxins from other microbial species, which have reduced toxicity butretained immunostimulatory capacity, such as modified heat-labile toxinfrom Gram-negative bacteria or staphylococcal enterotoxins may also beused to generate a similar effect. These molecules are particularlysuited to mucosal administration.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and advantageously contain 10-95% of activeingredient, preferably 25-70%.

Thus, an aspect of the present invention relates to an immunogeniccomposition, a pharmaceutical composition, or a vaccine as describedherein, which is formulated for intradermal, transdermal, subcutaneous,intramuscular or mucosal application.

The adjuvant is preferably selected from the group consisting ofdimethyloctadecylammonium bromide (DDA), dimethyloctadecenylammoniumbromide (DODAC), Quil A, poly I:C, aluminium hydroxide, Freund'sincomplete adjuvant, IFN-γ, IL-2, IL-12, monophosphoryl lipid A (MPL),Treholose Dimycolate (TDM), Trehalose Dibehenate and muramyl dipeptide(MDP).

The polypeptides may also be used for immunizing a mammal against ETECor treating the mammal against ETEC.

Therefore, one aspect of the present invention relates to a method forimmunizing a mammal, the method comprising administering to the mammalan immunogenic composition, a pharmaceutical composition or a vaccine asdescribed herein.

Another aspect of the present invention relates to a method for treatinga mammal, which is infected with ETEC comprising administering to themammal an immunogenic composition, a pharmaceutical composition or avaccine as described herein.

An embodiment of the present invention relates to a polypeptide, animmunogenic composition or a pharmaceutical composition for use asdescribed herein or a method as described herein, wherein the mammal isa human.

In another embodiment of the present invention is the mammal an animalselected from the group consisting of a pig, a cow, a sheep, and ahorse.

A further aspect of the present invention relates to a polypeptide, animmunogenic composition, a pharmaceutical composition, or a vaccine asdescribed herein for use in preventing or treating infection caused byETEC.

Yet another aspect of the present invention relates to a polypeptide, animmunogenic composition, a pharmaceutical, or a vaccine as describedherein for use in the preparation of a medicament for treating infectioncaused by ETEC.

Nucleic Acids

By the terms “nucleic acid fragment” and “nucleic acid sequence” areunderstood any nucleic acid molecule including DNA, RNA, LNA (lockednucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. Also included arenucleic acid molecules comprising non-naturally occurring nucleosides.The term includes nucleic acid molecules of any length e.g. from 10 to10000 nucleotides, depending on the use. When the nucleic acid moleculeis for use as a pharmaceutical, e.g. in DNA therapy, or for use in amethod for producing a polypeptide according to the invention, amolecule encoding at least one epitope is preferably used, having alength from about 18 to about 1000 nucleotides, the molecule beingoptionally inserted into a vector.

When the nucleic acid molecule is used as a probe, as a primer or inantisense therapy, a molecule having a length of 10-100 is preferablyused.

According to the invention, other molecule lengths can be used, forinstance a molecule having at least 12, 15, 21, 24, 27, 30, 33, 36, 39,42, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 nucleotides (ornucleotide derivatives), or a molecule having at most 10000, 5000, 4000,3000, 2000, 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30 or 20nucleotides (or nucleotide derivatives).

Thus, one aspect of the present invention relates to a nucleic acidsequence encoding a polypeptide as described herein.

Diagnosis

Immunodiagnostics are well suited for the detection of even the smallestof amounts of biochemical substances such as antibodies. Antibodiesspecific for a desired antigen can be conjugated with a radiolabel,fluorescent label, or color-forming enzyme and are used as a “probe” todetect it. Well known applications include pregnancy tests,immunoblotting, ELISA and immunohistochemical staining of microscopeslides. The speed, accuracy and simplicity of such tests has led to thedevelopment of rapid techniques for the diagnosis of disease.

Therefore, an aspect of the present invention relates to a polypeptide,an immunogenic composition, a pharmaceutical composition, or a vaccineas described herein for use in the diagnosis of an infection caused byETEC.

The polypeptide, immunogenic composition, or pharmaceutical compositionas described herein may also be used to detect the presence of ETEC in asample or used as an indication whether a sample or subject may containETEC.

It should be noted that embodiments and features described in thecontext of one of the aspects of the present invention also apply to theother aspects of the invention.

All patent and non-patent references cited in the present application,are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the followingnon-limiting examples.

EXAMPLES Example 1—BEMAP Method Results

BEMAP relies on β-elimination of O-linked carbohydrate modifications,Michael addition of 2-Aminoethyl phosphonic acid (AEP) and TiO₂enrichment of phosphopeptides. Thus, BEMAP combines a firmly establishedin vitro chemical modification with a highly selective enrichmentprotocol (Thingholm et al., 2006) and the reactions take place in asingle volume without the need for intermediate purification steps asdescribed in the Experimental Procedures section.

The BEMAP method was first established using a synthetic mannosylatedpeptide as a model compound. As shown in FIGS. 1A and 1B, MALDI MSdemonstrated 30 that BEMAP efficiently replaces the carbohydrate moietyof the synthetic peptide (m/z=1181.59 Da) with the AEP group and thusproduces a phosphopeptide (m/z=1126.64 Da).

The overall efficiency of substitution exceeds 95% (FIG. 1B) without theformation of degradation products. The AEP-modified peptide was thenstrongly enriched using affinity chromatography with TiO₂; both theintact glycopeptide and the β-eliminated peptide (1001.62 Da) wereabsent in the MALDI MS spectrum after enrichment (FIG. 1C).

The inventors found that BEMAP converts other glycopeptides into aphosphopeptide, independent of the identity of the linked monosaccharide(data not shown). It should be noted that the TiO₂ purification step ofBEMAP also targets phosphopeptides. Therefore, as a precaution theinventors use the enzyme Alkaline phosphatase to dephosphorylate anynative phosphopeptides which otherwise may result in false positiveidentifications.

The inventors analyzed the gas phase-induced fragmentation properties ofthe converted glycopeptide. As shown in FIG. 2, the exchange of acarbohydrate moiety with AEP has several advantages. The AEP additionsubstitutes a labile glycoside bond with a stronger covalent C—N bond,which greatly improves mapping of glycosylated residues by higher-energycollisional dissociation (HCD) fragmentation. Moreover, the AEP groupyielded two characteristic ions during HCD fragmentation (m/z=126.03 Daand m/z=138.03 Da), which are very useful for the identification offormerly glycosylated peptides in complex MS/MS spectra. It should benoted that the AEP molecule is constituted by a phosphonate functionalgroup, which is stable under CID and HCD fragmentation conditionscompared to the phosphate one, which is labile under these conditions.This allows unambiguous assignment of modified amino acid residues andavoids false positives in site localization assignment (data not shown).

Next, the inventors applied BEMAP to a purified heptosylated protein:Ag43 from E. coli (Knudsen et al., 2008). As may be seen in FIG. 1D,in-gel digestion of the glycosylated protein yielded heptosylated andunmodified peptides. Heptosylated peptides are marked by an asterisk.From the digested peptide mix, BEMAP enriched the three heptosylatedpeptides present in FIG. 1D as well as four additional glycopeptidesinitially undetectable by MALDI MS (FIGS. 1E and 1F). It is concludedthat BEMAP is a specific and sensitive method for detecting proteinglycosylation.

Results

The outer membrane protein fraction of H10407 was isolated and subjectedto BEMAP analysis for identification of glycoproteins. This approachidentified the protein YghJ, a putative lipoprotein AcfD homolog.

Discussion

BEMAP relies on nucleophile tagging using 2-Aminoethyl phosphonic acid(AEP) rather than e.g. DTT. BEMAP method selectivity is achieved withthe glycan-for-phosphate molecule exchange combined with a highlyspecific enrichment protocol for downstream sample processing (Thingholmet al., 2006). Importantly, the BEMAP chemistry can be applied inprinciple to any organism on a large-scale proteomics level irrespectiveof the chemical properties of the O-linked monosaccharide. Asdemonstrated in FIGS. 1A-F, BEMAP replaces the carbohydrate moiety of asynthetic glycosylated peptide with a phosphotag in a chemical reactionexceeding 95% efficiency. Moreover, HCD MS/MS fragmentation of enrichedBEMAP samples yields diagnostic ions instrumental for glycopeptide MS/MSspectrum identification as well as enabling unambiguous assignment ofthe modified amino acid residue, see FIGS. 2A-B.

To identify specific pathogenic E. coli associated glycoproteins ofpotential therapeutic value the inventors compared the outer membraneprotein complement to non-pathogenic reference strain MG1655 sampledunder identical conditions. By applying the BEMAP workflow, theinventors identified the ETEC vaccine candidate YghJ, a putativelipoprotein AcfD homolog. Based on analyses, the inventors propose thatnovel vaccines directed against ETEC should not only be selected amongstthe glycoproteins expressed by the pathogen but can in principle also betargeting glycosylated domains of proteins which otherwise share 100%identity among E. coli strains.

Experimental Procedures:

Lyophilized peptide sample is resuspended in 100 μl BEMAP solutionconsisting of 0.4 M 2-AEP (Sigma; 268674), 0.75 M NaOH (Sigma; S8045),20 mM Ba(OH)₂ (Sigma; 433373) and incubate at 37° C. in a heating blockfor 3.15 hours shaking at 1300 r.p.m. The reaction is stopped byacidification (1% TFA final concentration). Sample volume is increasedto 1 ml and the peptides are purified on an Oasis HLB Plus shortcartridge (Waters) as recommend by manufacturer and subsequentlylyophilized. TiO₂ enrichment was performed as described by Tingholm etal., 2006.

ETEC H10407 lead molecule: Putative lipoproteinAcfD homolog YghJ, ETEC 3241, CBJ02741Primary sequence of YghJ (SEQ ID NO: 1):MNKKFKYKKS LLAAILSATL LAGCDGGGSG SSSDTPPVDSGTGSLPEVKP DPTPNPEPTP EPTPDPEPTP EPIPDPEPTPEPEPEPVPTK TGYLTLGGSQ RVTGATCNGE SSDGFTFKPGEDVTCVAGNT TIATFNTQSE AARSLRAVEK VSFSLEDAQELAGSDDKKSN VSLVTSSNS CPANTEQVCL TFSSVIESKRFDSLYKQIDL APEEFKKLVN EEVENNAATD KAPSTHTSPVVPVTTPGTKP DLNASFVSAN AEQFYQYQPT EIILSEGRLVDSQGYGVAGV YYTNSGRGV TGENGEFSFS WGEAISFGIDTFELGSVRGN KSTIALTELG DEVRGANIDQ LIHRYSTTGQNNTRVVPDDV RKVFAEYPNV INEIINLSLS NGATLGEGEQVVNLPNEFIE QFNTGQAKEI DTAICAKTDG CNEARWFSLTTRNVNDGQIQ GVINKLWGVD TNYKSVSKFH VFHDSTNFYGSTGNARGQAV VNISNAAFPI LMARNDKNYW LAFGEKRAWDKNELAYITEA PSIVRPENVT RETASFNLPF ISLGQVGDGKLMVIGNPHYN SILRCPNGYS WNGGVNKDGQ CTLNSDPDDMKNFMENVLRY LSNDRWLPDA KSSMTVGTNL ETVYFKKHGQVLGNSAPFAF HKDFTGITVK PMTSYGNLNP DEVPLLILNGFEYVTQWGSD PYSIPLRADT SKPKLTQQDV TDLIAYMNKGGSVLIMENVM SNLKEESASG FVRLLDAAGL SMALNKSVVNNDPQGYPDRV RQRRSTPIWV YERYPAVDGK PPYTIDDTTKEVIWKYQQEN KPDDKPKLEV ASWQEEVEGK QVTQFAFIDEADHKTPESLA AAKQRILDAF PGLEVCKDSD YHYEVNCLEYRPGTDVPVTG GMYVPQYTQL DLSADTAKAM LQAADLGTNIQRLYQHELYF RTNGRQGERL NSVDLERLYQ NMSVWLWNETKYRYEEGKED ELGFKTFTEF LNCYTNNAYV GTQCSAELKKSLIDNKMIYG EESSKAGMMN PSYPLNYMEK PLTRLMLGRSWWDLNIKVDV EKYPGVVNTN GETVTQNINL YSAPTKWFAGNMQSTGLWAP AQQEVSIESK STVPVTVTVA LADDLTGREKHEVSLNRPPR VTKTYDLKAN DKVTFKVPYG GLIYIKGDSKEVQSADFTFT GVVKAPFYKD GKWQHDLNSP APLGELESASFVYTTPKKNL NASNYTGGLE QFANDLDTFA SSMNDFYGRDSEDGKHRMFT YKNLPGHKHR FANDVQISIG DAHSGYPVMNSSFSPNSTTL PTTPLNDWLI WHEVGHNAAE TPLTVPGATEVANNVLALYM QDRYLGKMNR VADDITVAPE YLEESNGQAWARGGAGDRLL MYAQLKEWAE KNFDIKKWYP DGTPLPEFYSEREGMKGWNL FQLMHRKARG DEVSNDKFGG KNYCAESNGNAADTLMLCAS WVAQTDLSEF FKKWNPGANA YQLPGASEMSFEGGVSQSAY NTLASLDLPK PEQGPETINQ VTEHKMSAE

Unique to H10407 Compared to Other E. coli

TABLE 1Identified Tryptic glycopeptides using BEMAP combined with ESI-MS/MS(SEQ ID NOs: 2-7 and SEQ ID NO: 24-50; glycosylations in bold): Mod ModMod Mod Mod Mod Mod Start End Seq AA #1 AA #2 AA #3 AA #4 AA #5 AA #6AA #7 355 364 YSTTGQNN S356 1357 1358 1363 TR (SEQ ID NO: 2) 609 615YLSNDR S612 (SEQ ID NO: 3) 588 609 DGQCTLNS S595 DPDDMKNF MENVLR (SEQ IDNO: 4) 1341 1362 VADDITVAP S1355 EYLEESNGQ AWAR (SEQ ID NO: 5) 1048 1076VDVEKYPGV T1059 T1063 T1065 VNTNGETVT QNINLYSAP TK (SEQ ID NO: 6) 102143 VTGATCNG T103 T106 S111 S112 T116 T124 T130 ESSDGFTFK PGEDVTCVAGNTTIATFN TQSEAAR (SEQ ID NO: 7) 91 101 TGYLTLGGS S99 QR (SEQ ID NO: 24)144 167 SLRAVEKVS S144 FSLEDAQEL AGSDDK (SEQ ID NO: 25) 169 199SNAVSLVTS S177 S180 T185 T191 SNSCPANTE QVCLTFSSV IESK (SEQ ID NO: 26)218 231 LVNEEVENN T229 AATDK(SEQ ID NO: 27) 419 435 EIDTAICAKT T422DGCNEAR (SEQ ID NO: 28) 428 442 TDGCNEAR T428 S438 T440 T441 WFSLTTR(SEQ ID NO: 29) 456 468 LWGVDTNY S467 KSVSK (SEQ ID NO: 30) 469 486FHVFHDSTN S476 FYGSTGNAR (SEQ ID NO: 31) 487 504 GQAVVNISN S494AAFPILMAR (SEQ ID NO: 32) 622 636 SSMTVGTN S622 S623 T625 T632 LETVYFK(SEQ ID NO: 33) 705 719 LTQQDVTDL T711 IAYMNK (SEQ ID NO: 34) 720 734GGSVLIMEN S722 VMSNLK (SEQ ID NO: 35) 735 743 EESASGFVR S739 (SEQ IDNO: 36) 757 769 SVVNNDPQ S757 GYPDR (SEQ ID NO: 37) 818 830 LEVASWQEES822 VEGK (SEQ ID NO: 38) 845 853 TPESLAAAK S848 (SEQ ID NO: 39) 909 922AMLQAADL T918 GTNIQR (SEQ ID NO: 40) 923 935 LYQHELYFR T932 TNGR (SEQID NO: 41) 936 947 QGERLNSV S942 DLER (SEQ ID NO: 42) 948 963 LYQNMSVWT960 LWNETKYR (SEQ ID NO: 43) 1000 1015 KSLIDNKMI S1001 S1013 S1014YGEESSK (SEQ ID NO: 44) 1016 1034 AGMMNPSY S1022 PLNYMEKPL TR (SEQ IDNO: 45) 1101 1120 STVPVTVTV T1102 T1106 T1116 ALADDLTGR EK (SEQ IDNO: 46) 1134 1142 TYDLKANDK T1134 (SEQ ID NO: 47) 1134 1146 TYDLKANDKT1144 VTFK (SEQ ID NO: 48) 1143 1160 VTFKVPYGG S1159 LIYIKGDSK (SEQ IDNO: 49) 1418 1431 ARGDEVSN S1424 DKFGGK (SEQ ID NO: 50)

Identified YghJ glycopeptides listed as probableepitopes presented by Antigen Presenting Cells(SEQ ID NOs: 8-23, glycosylations in bold): QLIHRYSTTGQNN IHRYSTTGQNNTRTTGQNNTRVVPDD NVLRYLSNDRWLP GQCTLNSDPDDMK PEYLEESNGQAWA YPGVVNTNGETVTVNTNGETVTQNIN TNGETVTQNINLY GGSQRVTGATCNG QRVTGATCNGESS ATCNGESSDGFTFTCNGESSDGFTFK ESSDGFTFKPGED KPGEDVTCVAGNT TCVAGNTTIATFN

Example 2—Immunogenicity of ETEC Glycosylated Proteins

An immunogenic polypeptide is defined as a polypeptide that induces animmune response.

The immune response may be monitored by one of the following methods:

An in vitro cellular response is determined by release of a relevantcytokine such as IFN-γ, from lymphocytes withdrawn from an animal orhuman currently or previously infected with ETEC, or by detection ofproliferation of these T cells. The induction is performed by additionof the polypeptide or the immunogenic part to a suspension comprisingfrom 1×10⁵ cells to 3×10⁵ cells per well. The cells are isolated fromeither blood, the spleen, the liver or the lung and the addition of thepolypeptide or the immunogenic part of the polypeptide result in aconcentration of not more than 20 μg per ml suspension and thestimulation is performed from two to five days. For monitoring cellproliferation, the cells are pulsed with radioactive labeled Thymidineand after 16-22 hours of incubation, the proliferation is detected byliquid scintillation counting. A positive response is a response morethan background plus two standard deviations. The release of IFN-γ canbe determined by the ELISA method, which is well known to a personskilled in the art. A positive response is a response more thanbackground plus two standard deviations. Other cytokines than IFN-γcould be relevant when monitoring an immunological response to thepolypeptide, such as IL-12, TNF-α, IL-4, IL-5, IL-10, IL-6, TGF-β.

Another and more sensitive method for determining the presence of acytokine (e.g. IFN-γ) is the ELISPOT method where the cells isolatedfrom either the blood, the spleen, the liver or the lung are diluted toa concentration of preferable of 1 to 4×10⁶ cells/ml and incubated for18-22 hrs in the presence of the polypeptide or the immunogenic part ofthe polypeptide resulting in a concentration of not more than 20 μg perml.

The cell suspensions are hereafter diluted to 1 to 2×10⁶/ml andtransferred to Maxisorp plates coated with anti-IFN-γ and incubated forpreferably 4 to 16 hours. The IFN-γ producing cells are determined bythe use of labelled secondary anti-IFN-antibody and a relevant substrategiving rise to spots, which can be enumerated using a dissectionmicroscope. It is also a possibility to determine the presence of mRNAcoding for the relevant cytokine by the use of the PCR technique.Usually one or more cytokines will be measured utilizing for example thePCR, ELISPOT or ELISA. It will be appreciated by a person skilled in theart that a significant increase or decrease in the amount of any ofthese cytokines induced by a specific polypeptide can be used inevaluation of the immunological activity of the polypeptide.

An in vitro cellular response may also be determined by the use of Tcell lines derived from an immune individual or an ETEC infected personwhere the T cell lines have been driven with either live ETEC, extractsfrom the bacterial cell or culture filtrate for 10 to 20 days with theaddition of IL-2. The induction is performed by addition of not morethan 20 μg polypeptide per ml suspension to the T cell lines containingfrom 1×10⁵ cells to 3×10⁵ cells per well and incubation is performedfrom two to six days. The induction of IFN-γ or release of anotherrelevant cytokine is detected by ELISA. The stimulation of T cells canalso be monitored by detecting cell proliferation using radioactivelylabeled Thymidine as described above. For both assays, a positiveresponse is a response more than background plus two standarddeviations.

An in vivo cellular response may be determined as a positive DTHresponse after intradermal injection or local application patch of atmost 100 μg of the polypeptide or the immunogenic part to an individualwho is clinically or subclinically infected with ETEC, a positiveresponse having a diameter of at least 5 mm 72-96 hours after theinjection or application.

An in vitro humoral response is determined by a specific antibodyresponse in an immune or infected individual. The presence of antibodiesmay be determined by an ELISA technique or a Western blot where thepolypeptide or the immunogenic part is absorbed to either anitrocellulose membrane or a polystyrene surface. The serum ispreferably diluted in PBS from 1:10 to 1:100 and added to the absorbedpolypeptide and the incubation being performed from 1 to 12 hours. Bythe use of labeled secondary antibodies the presence of specificantibodies can be determined by measuring the presence or absence of aspecific label e.g. by ELISA where a positive response is a response ofmore than background plus two standard deviations or alternatively avisual response in a Western blot.

Another relevant parameter is measurement of the protection in animalmodels induced after vaccination with the polypeptide in an adjuvant orafter DNA vaccination. Suitable animal models include primates, guineapigs or mice, which are challenged with an infection of an ETEC. Readoutfor induced protection could be decrease of the bacterial load in targetorgans compared to non-vaccinated animals, prolonged survival timescompared to non-vaccinated animals and diminished weight loss orpathology compared to non-vaccinated animals.

The glycosylated polypeptides described herein are immunogenic when oneof the above-described tests is positive.

Example 3—Schematic Overview of Assays and Experiments Used toCharacterize Glycosylated as Well as Non-Glycosylated YghJ ProteinProperties

TABLE 2 Type of experiment Mouse challenge Serum and mucosal antibodyresponses Antibody mediated inhibition of ETEC binding to Caco-2Antibody mediated inhibition of ETEC binding to Caco-2; cAMP releasemeasurement Degradation of intestinal mucin MUC3 Quantitative YghJ −MUC3 interaction assessment Degradation of intestinal mucin MUC2

An overview of the assays used for testing a wide variety of YghJfeatures is given in Table 2.

Example 4—Vaccination with Glycosylated YghJ Affords Better ProtectionAgainst Intestinal Colonization of ETEC in Mice Compared to theNon-Glycosylated Protein Versions

Assay Type: Mouse Challenge Studies

Materials and Methods:

Seven groups of CD-1 mice were immunized with either adjuvant only(control), or appropriate amount of adjuvant+25 μg of glycosylated YghJor adjuvant+e.g. 25 μg of non-glycosylated YghJ on days 0, 14, 28. Onday 40, mice were treated with streptomycin [e.g. 5 g per liter] indrinking water for 24 hours, followed by drinking water alone for 18hours. After administration of famotidine to reduce gastric acidity,mice were challenged with 106 cfu of a chloramphenicol-resistant ETECstrain by oral gavage. Fecal samples (6 pellets/mouse) were collected onday 42 before oral gavage, re-suspended in buffer (10 mM Tris, 100 mMNaCl, 0.05% Tween 20, 5 mM Sodium Azide, pH 7.4) overnight at 4° C.,centrifuged to pellet insoluble material, and recover supernatant forfecal antibody testing (below). Twenty-four hours after infection, micewere sacrificed, sera were collected, and dilutions of saponinsmall-intestinal lysates were plated onto Luria agar plates containingchloramphenicol (40 μg/ml).

Experimental outcome: As determined by CFU counting, fecal samples frommice immunized with glycosylated antigen YghJ contained fewer ETECcompared to fecal samples from mice immunized with non-glycosylatedantigen versions.

Example 5—Immunization with Glycosylated Antigen YghJ Generates RobustSerum and Mucosal Antibody Responses

Assay Type: ELISA Assay Probing Relative Levels of IgA, IgM and IgG

Materials and Methods:

Murine immune responses to adjuvant, glycosylated and non-glycosylatedversions of YghJ were determined using ELISA. Briefly, ELISA wells wereincubated at 4° C. overnight with proteins at a final concentration of 4μg/ml in 0.1 M NaHCO3 buffer (pH 8.6), washed the following day withTris-buffered saline containing 0.005% Tween 20 (TBS-T), and blockedwith 1% bovine serum albumin (BSA) in TBS-T for 1 h at 37° C. prior tothe addition of the samples. Sera was serial diluted in TBS-T with 1%BSA, and 100 μl was added to each ELISA well, followed by incubation at37° C. for 1 h. After three washes with TBS-T, horseradishperoxidase-conjugated secondary antibody (either goat anti-mouse IgA,IgM, or IgG) was added at a final dilution of 1:5,000, followed byincubation for an additional hour before washing and development withTMB (3,3′,5,5′-tetramethylbenzidine)-peroxidase substrate (KPL). KineticELISA data are expressed as Vmax in milliunits/min.

Experimental outcome: Immunization with glycosylated antigen YghJgenerates robust IgA, IgG and IgM antibody responses as compared tonon-glycosylated versions

Example 6—Monoclonal Antibodies Raised Against Glycosylated YghJInhibits ETEC Binding to Intestinal Epithelial Cells to a Higher ExtentCompared Monoclonal Antibodies Raised Against Non-Glycosylated YghJProtein Version

Assay Type: Adhesion Assay

Materials and Methods:

In vitro, Caco-2 epithelial cell monolayers were infected with ETECH10407 at multiplicities of infection of approximately 100(bacteria/cell). Cultures of bacteria were grown overnight in Luriabroth from frozen glycerol stocks, diluted 1:100, and grown for 1 h. Onemicroliter of bacterial culture is added to confluent Caco-2 monolayersseeded into 96-well plates preincubated with or without antibodies. Twohours after inoculation, the monolayers were washed 3 times with tissueculture medium after which bacteria were isolated, serial diluted andplated to count CFU the following day.

Experimental outcome: Monoclonal antibodies raised against glycosylatedYghJ inhibits ETEC binding to intestinal epithelial cells to a higherextent compared monoclonal antibodies raised against non-glycosylatedYghJ protein version.

Example 7—Monoclonal Antibodies Raised Against Glycosylated YghJInhibits ETEC Binding to Intestinal Epithelial Cells to a Higher ExtentCompared to Monoclonal Antibodies Raised Against Non-Glycosylated YghJProtein Version

Assay Type: Adhesion Assay Coupled to cAMP Enzyme Immunoassay

Materials and Methods:

In vitro, Caco-2 epithelial cell monolayers were infected with ETECH10407 at multiplicities of infection of approximately 100(bacteria/cell). Cultures of bacteria were grown overnight in Luriabroth from frozen glycerol stocks, diluted 1:100, and grown for 1 h. Onemicroliter of bacterial culture is added to confluent Caco-2 monolayersseeded into 96-well plates preincubated with or without antibodies. Twohours after inoculation, the monolayers were washed 3 times with tissueculture medium, and the medium was replaced with 100 μl of freshmedium/well and returned to the incubator (37° C., 5% CO₂) for 2.5 h.Subsequently, cyclic AMP (cAMP) enzyme immunoassay (EIA) (Arbor Assays,Ann Arbor, Mich.) was used to examine the efficiency of toxin delivery.

Experimental outcome: Addition of antibodies raised against glycosylatedYghJ results in lower levels of released cAMP into the growth mediumcompared to monoclonal antibodies raised against non-glycosylated YghJprotein version.

Example 8—Glycosylated YghJ Degrade Intestinal Mucin MUC3 in aDose-Dependent Fashion to a Higher Extent Compared to theNon-Glycosylated YghJ Protein Version and Mucin Degrading Activity canbe Blocked with Monoclonal Antibodies Targeting Glycosylated Epitopes

Assay Type: Western Blot

Materials and Methods:

To examine the activity of glycosylated and non-glycosylated YghJagainst the cell-associated mucin MUC3, Caco-2 epithelial cells weregrown in monolayers in 96-well tissue culture plates for 48 to 72 hpostconfluence to optimize MUC3 expression on the epithelial surface.Supernatant was removed and replaced with 100 μl of minimum essentialmedium (MEM) containing YghJ (+/−glycosylation; final concentration of1-500 μg/ml) either with or without aliquots of antibody. Followingovernight treatment of the cell monolayers at 37° C. and 5% CO₂, themedium was removed, and the monolayers were lysed in 20 μl of lysisbuffer (e.g. 50 mM sodium phosphate, 250 mM NaCl, 0.1% Triton X-100, 0.1mM phenylmethylsulfonyl fluoride [PMSF], and complete EDTA-free proteaseinhibitor cocktail [Roche]). Following incubation on ice for 30 min andrepeated freeze (dry ice)-thaw (37° C.) cycles, the lysates werecentrifuged at 10,000×g (4° C.) to pellet debris. Clarified lysates werethen separated on gradient (3 to 8% Tris-acetate; Invitrogen) PAGE.Following transfer to nitrocellulose membranes, Caco-2 lysates wereimmunoblotted with anti-MUC3A/B goat polyclonal IgG antibodies (F-19[catalog no. sc-13314; Santa Cruz]) that recognize an internal region ofmucin 3A of human origin (gene identification [ID] 4584).

Experimental outcome: As determined by Western blotting, Caco-2 cellsexposed to glycosylated YghJ displays higher extent of MUC3 degradationcompared to cells incubated with the non-glycosylated protein variant.Moreover, the proteolytic activity of YghJ can be blocked by addingmonoclonal antibodies targeting the glycosylated amino acids.

Example 9—Glycosylated YghJ Interacts Stronger with the Human IntestinalMucin, MUC3, Compared to the Non-Glycosylated YghJ Protein Version

Assay Type: Far Western Blot

Materials and Methods

To examine interaction of YghJ with the human intestinal mucin MUC3,lysate from Caco-2 cells containing MUC3 was separated by SDS-PAGE asdescribed above and transferred to nitrocellulose membranes. To examineinteraction with MUC3, purified protein was spotted on nitrocellulosemembranes. Far Western analysis was then performed with purified YghJ3×FLAG. Briefly, nitrocellulose membranes with immobilized mucins wereblocked for 1 h with 1% bovine serum albumin (BSA) in PBS beforeincubating with 50 μg/ml of purified YghJ (+/−glycosylations) overnightat 4° C. Proteins were detected by immunoblotting using antimucinantibodies or anti-YghJ monoclonal antibody obtained from mice.

Expected outcome: When exposing immobilized MUC3 to either glycosylatedYghJ or the non-glycosylated protein variant, Far Western blotting showsthat the modified YghJ exhibits stronger binding towards the mucin.

Example 10—Glycosylated YghJ Degrade Purified Intestinal Mucin MUC2 in aDose-Dependent Fashion to a Higher Extent Compared to theNon-Glycosylated YghJ Protein Version and Mucin Degrading Activity canbe Blocked with Affinity Purified Antibodies

Assay Type: Western Blot

Materials and Methods

MUC2 was purified from supernatants of tissue culture medium from LS174Tcells (ATCC CL-188), a goblet cell-like adenocarcinoma line that makesabundant MUC2. Briefly, LS174T cells were grown as described above;conditioned medium was recovered, concentrated by ultrafiltration usinga 100-kDa-molecular-weight-cutoff filter (MWCO), and then bufferexchanged with 10 mM Tris-HCl and 250 mM NaCl (pH 7.4) prior to sizeexclusion chromatography using Sepharose CL-2B resin. Fractions werechecked for MUC2 by anti-MUC2 dot immunoblotting. MUC2-positivefractions, corresponding to a protein peak in the column void volume,were separated on 3 to 8% Tris-acetate gradient gels, stained with SyproRuby to check purity, and immunoblotted using anti-MUC2 to verify theidentity of the protein. Fractions containing intact, full-length MUC2were then pooled and saved at −80° C. for subsequent assays.

To examine degradation of purified MUC2, 0.1 μg of protein was treatedfor at least 30 min with 5 μg of either glycosylated or non-glycosylatedYghJ at 37° C. Affinity purified antibodies, isolated from rat exposedto either the glycosylated or non-glycosylated antigen, was added toreaction mixture in order to inhibit MUC2 degradation. Reaction productswere resolved by SDS-PAGE or agarose gels optimized for proteinseparation, and MUC2 digests were examined with anti-MUC2 rabbitpolyclonal (IgG) (H-300 [catalog no. sc-15334; Santa Cruz]) thatrecognizes an epitope corresponding to amino acids 4880 to 5179 at the Cterminus of human mucin 2 (gene ID 4583).

Expected outcome: The degradation rate of purified intestinal mucin MUC2is higher when exposed to glycosylated YghJ as compared tonon-glycosylated YghJ. Furthermore, mucin degradation can be blockedwith affinity-purified YghJ antibodies.

REFERENCES

-   Thingholm et al. (2006), Nat. Protoc. 1, 1929-1935-   Knudsen et al. (2008), Biochem. J. 412, 563-577-   Chou and Schwartz (2011), Curr. Protoc. Bioinformatics, chapter 13,    15-24-   Pearson and Lipman (1988), Proc. Natl. Acad. Sci. 85, 2444-2448.-   Thompson et al. (1994), Nucleic Acids Res. 11, 4673-4680.

1. A polypeptide comprising: a) SEQ ID NO: 1, b) a polypeptide having atleast 75% sequence identity to the full length sequence of SEQ ID NO: 1,or c) a polypeptide fragment of SEQ ID NO: 1 comprising at least 5 aminoacids and having at least 75% sequence identity to a segment of SEQ IDNO: 1, said segment of SEQ ID NO:1 having the same number of amino acidsas said polypeptide fragment, wherein the polypeptide is glycosylated inat least one position.